(Not Applicable)
The present invention relates to regulatory elements that are linked to a gene involved in apoptosis. The invention further relates to methods for identifying agents that modulate expression of a gene involved in apoptosis.
Apoptosis, or programmed cell death, is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Recent studies of apoptosis have implied that a common metabolic pathway leading to cell death may be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation and infection by human immunodeficiency virus (HIV). Wyllie (1980) Nature 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res. Commun. 118:392-399; Duke and Cohen (1986) Lymphokine Res. 5:289-299; Tomei et al. (1988) Biochem. Biophys. Res. Commun. 155:324-331; Kruman et al. (1991) J. Cell. Physiol. 148:267-273; Ameisen and Capron (1991) Immunology Today 12:102; and Sheppard and Ascher (1992) J. AIDS 5:143. Agents that modulate the biological control of apoptosis thus have therapeutic utility in a wide variety of conditions.
Apoptotic cell death is characterized by cellular shrinkage, chromatin condensation, cytoplasmic blebbing, increased membrane permeability and interchromosomal DNA cleavage. Kerr et al. (1992) FASEB J. 6:2450; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267. The blebs, small, membrane-encapsulated spheres that pinch off of the surface of apoptotic cells, may continue to produce superoxide radicals which damage surrounding cell tissue and may be involved in inflammatory processes.
While apoptosis is a normal cellular event, it can also be induced by pathological conditions and a variety of injuries. Apoptosis is involved in a wide variety of conditions including but not limited to, cardiovascular disease; cancer regression; immune disorders, including but not limited to systemic lupus erythematosus; viral diseases; anemia; neurological disorders; diabetes; hair loss; rejection of organ transplants; prostate hypertrophy; obesity; ocular disorders; stress; aging; and gastrointestinal disorders, including but not limited to, diarrhea and dysentery.
In Alzheimer""s disease, Parkinson""s disease, Huntington""s chorea, epilepsy, amyotrophic lateral sclerosis, stroke, ischemic heart disease, spinal cord injury and many viral infections, for example, abnormally high levels of cell death occur. In at least some of these diseases, there is evidence that the excessive cell death occurs through mechanisms consistent with apoptosis. Among these are 1) spinal cord injury, where the severing of axons deprives neurons of neurotrophic factors necessary to sustain cellular viability; 2) stroke, where after an initial phase of necrotic cell death due to ischemia, the rupture of dead cells releases excitatory neurotransmitters such as glutamate and oxygen free radicals that stimulate apoptosis in neighboring healthy neurons; and 3) Human Immunodeficiency Virus (HIV) infection, which induces apoptosis of T-lymphocytes.
In contrast, the level of apoptosis is decreased to abnormal levels in cancer cells, which allows the cancer cells to survive longer than their normal cell counterparts. As a result of the increased number of surviving cancer cells, the mass of a tumor can increase even if the doubling time of the cancer cells does not increase. Furthermore, the high level of expression in a cancer cell of the bcl-2 gene, which is involved in regulating apoptosis and, in some cases, necrotic cell death, renders the cancer cell relatively resistant to chemotherapeutic agents and to radiation therapy.
It is convenient to divide the process of physiological cell death into phases. Vaux and Strasser (1996) Proc. Natl. Acad. Sci. 93:2239-2244. The earliest phase is the stimulus that provokes the apoptotic response. This may be an external signal delivered through surface receptors or may originate inside the cell from the action of a drug, toxin, or radiation. The next phase includes detection of this signal or metabolic state and transduction of the signal. Signal transduction pathways send this message to the cell death effector machinery. The effector phase is the third part of the cell death mechanism and includes the proteases that are activated during apoptosis, as well as their positive and negative regulators. The fourth phase of cell death is the postmortem phase, in which the cell""s chromatin condenses and its DNA is degraded.
The activation or signaling phase of cell death encompasses a great variety of signal transduction pathways that mediate signals from outside the cell, as well as others that originate inside the cell. Two members of the TNF superfamily of receptors, TNFR 1 and CD95, when bound to their respective ligands, TNF-I and CD95L (FasL) can rapidly transduce an apoptotic cell death signal (6-8). Nagata and Golstein (1995) Science 267:1449-1456; Cleveland and Ihle (1995) Cell 81:479-482; Schutze-Osthoff (1994) Trends Cell Biol. 4:421-426. Cell death induced by the CD95/CD95L system is important for the elimination of potentially autoreactive peripheral T cells and contributes to T cell-mediated cytoxicity, whereas the TNFR I/TNF-I system plays a critical role in host defense against microorganisms and their pathogenic factors.
In recent years, a family of proteins has been discovered that controls apoptosis. The prototype of this family is Bcl-2, a protein that inhibits most types of apoptotic cell death and is thought to function by regulating an antioxidant pathway at sites of free radical generation. Hockenbery et al. (1993) Cell 75:241-251. Together, the Bcl-2 family of proteins are important intracellular modulators of apoptosis and can be divided into two groups based on their effect on apoptosis. Thus, in a general sense, Bcl-2, Bcl-xL, Mcl-1, BHRF-1 and E1B19K are cell death inhibitors (anti-apoptotic), while Bak, Bax and Bcl-xS accelerate cell death (pro-apoptotic).
Bcl-2 family members are generally localized to the outer mitochondrial membrane, the nuclear membrane and the endoplasmic reticulum, where they associate with membranes by virtue of their C-terminal hydrophobic tail. All members of the family have two highly conserved regions, called BH1 and BH2, that permit specific interactions between two members to form stable dimers. Their mechanism of action is presently unclear; however, it is known that the ratio of anti-apoptotic to pro-apoptotic Bcl-2 family members in a cell is critical to the cell""s survival following initiation of an apoptotic signal.
Bak is a member of the Bcl-2 family and is expressed in heart and other tissues. Bak protein is capable of either killing cells, or actively protecting cells from cell death, depending on how this protein interacts with other cellular proteins. Bcl-2 family members are extremely important in determining the fate of a cell following an apoptotic signal, and Bak may be the most important in the major organs such as heart. In the treatment of heart disease, viral infection and cancer, modulation of the expression of genes encoding proteins that control apoptosis is a major focal point.
Interferons (IFN) were originally discovered in the late 1950s as substances produced in animals infected with viruses that could elicit protection against subsequent viral infection. Isaacs and Lindenmann (1957) Proc. Royal Soc. Lond. (Biol.) 147:258-267. This activity was shown to reside in a group of functionally related polypeptides, IFNxcex1, -xcex2 and -xcex3, factors which were further discovered to possess a broad range of biological activities in addition to their antiviral action. IFNxcex1 and -xcex2, collectively known as the type I IFN, are synthesized by almost any nucleated cell in response to viral infection and function by binding to common receptors on the surfaces of target cells. IFNxcex3, referred to as type II IFN, is structurally unrelated to IFNxcex1 or -xcex2, is synthesized specifically by activated T cells and natural killer cells, and recognizes cell-surface receptors distinct from those recognized by the type I IFN. Despite their different structures and receptor-binding activities, the type I and type II IFN function in a very similar manner to influence a broad range of biological functions including the modulation of the immune response, inflammation, hematopoiesis, cell proliferation and differentiation. DeMaeyer and DeMaeyer-Guignard (1988) Interferons and Other Regulatory Cytokines. Wiley, N.Y.
The diverse effects of IFN are mediated by their binding to specific cell-surface receptors, activation of signal-transducing molecules and the consequent modulation of gene expression. The type I and type II IFNs produce distinct, though partially overlapping, effects on cells. The initial transmission of the IFN signal to the nucleus involves proteins that function in cooperation with one another. These include the Stat (signal transducer and activator of transcription) proteins and ISGF3xcex3 (IFN-stimulated gene factor 3xcex3 polypeptide).
In response to type I IFN, the 113 kDa protein Stat2, the 91 kDa protein Stat1xcex1 and the 84 kDa Stat1xcex2 become tyrosine phosphorylated. These proteins combine with the 48 kDa ISGF3xcex3 to form IFN-stimulated gene factor 3 (ISGF3), a multimeric complex that translocates to the nucleus and displays a distinct DNA-binding specificity for the IFN-stimulated response element (ISRE) found in the promoters of IFNxcex1-stimulated genes. Darnell et al. (1994) Science 264:1415-1420.
In contrast, IFNxcex3-inducible genes are activated when Stat1xcex1 becomes tyrosine phosphorylated and forms homodimers, termed GAF for IFNxcex3-activated factor, which are capable of binding to the IFNxcex3-responsive element termed the gamma-activated site (GAS). The two complexes, ISGF3 and GAF, recognize different sequences in the promoters of type I and type II IFN, respectively and are integral components of the system by which IFN stimulation received at the cell surface is translated into changes in gene transcription in the nucleus.
While the ISGF-3 and GAF are responsible for the initial transmission of the IFN signal to the nucleus, the proper regulation of the broad range of genes induced by the interferons involves other transcription factors as well. These include the IFN regulatory factors, or IRFs.
IRF-1, IRF-2 and IRF-3 have been identified as DNA-binding factors that function as regulators of both type I and type II inducible genes. These transcription factors are structurally related, particularly in their N-terminal regions that confer DNA binding specificity. The IRF also show significant amino acid sequence identity to ISGF3xcex3, the DNA binding component of the complex that recognizes ISRE in IFN-inducible genes and to ICSBP, which is expressed only in cells of lymphoid origin and which binds the ISRE of IFN-inducible genes in these cells. Au et al. (1995) Proc. Natl. Acad. Sci. 92:11657-11661. In addition, both IRF-1 and IRF-2 bind to the same sequence within the promoters of IFN-xcex1 and IFN-xcex2 genes. Harada et al. (1989) Cell 58:729-739. The IRF and ISGF3 have also been shown to bind to overlapping sequences in the promoters of many IFNxcex1/xcex2-inducible genes. Tanaka et al. (1993) Mol. Cell. Biol. 13:4531-4538. IRF-1 functions as an activator of interferon transcription, while IRF-2 binds to the same cis elements and represses IRF-1 action. IRF-1 and IRF-2 have been reported to act in a mutually antagonsitic manner in regulating cell growth: overexpression of the repressor IFR-2 leads to cell transformation, while concomitant overexpression of IRF-1 causes reversion. In addition to being a regulator of cell growth, IRF-1 is also a key transcription factor in the regulation of apoptosis. Taniguchi et al. (1995) J. Cancer Res. Clin. Oncol. 121:516-520; Tamura et al. (1995) Nature 376:596-599; Tanaka et al. (1994) Cell 77:829-839.
When normal embryo fibroblasts expressing activated c-H-ras were cultured in low serum or treated by anticancer drugs or ionizing radiation they were observed to lose viability by a process characteristic of apoptosis. In contrast, when IRF-1-/-fibroblasts expressing activated c-H-ras were subjected to the same treatment, the cells survived. Tanaka et al. (1994) Cell 77:829-839. The tumor suppressor p53 has been shown to regulate apoptosis in thymocytes, while in mitogen-activated mature T lymphocytes, DNA-damage-induced apoptosis was found to be dependent on IRF-1. Tamura et al. (1995) Nature 376:596-599. Clinical studies indicate that IRF-1 may function as an anti-oncogene in vivo, preventing the development of some forms of human leukemia. In a study of 13 patients with leukemia or myelodysplastic syndrome who exhibited cytogenetic abnormalities in the 5q31.1 chromosomal region, IRF-1 was the only gene found to be consistently deleted or rearranged in either or both alleles. Willman et al. (1993) Science 259:968-971. Splicing aberrations in the IRF-1 gene also occur at high frequency in patients with leukemia or myelodysplastic syndrome. Harada et al. (1993) Oncogene 9:3313-3320.
Treatment of cells with IFN-xcex3 can render them susceptible to apoptotic stimuli. For example, various cell lines display an increased sensitivity to cytotoxic signalling through TNFR 1 or CD95 following treatment with IFNxcex3. Yonehara et al. (1989) J. Exp. Med. 169:1747-1756; Fransen et al. (1986) Eur. J. Cancer and Clin. Oncol. 22:419-426; Tsujimoto et al. (1986) J. Immunol. 136:2441-2444. The human colon adenocarcinoma cell line, HT29, is particularly responsive to IFNxcex3, which markedly increases its sensitivity to TNF-I as well as anti-Fas antibodies (Ab) mediated cytotoxicity. The IFN-xcex3-induced sensitivity of HT29 cells to TNF-I or anti-Fas Ab mediated cell death has been attributed to the upregulation of CD95 and TNFR 1, which also occurs during IFN-xcex3 treatment. However, since TNF and anti-Fas Ab induce cell death by apoptotic mechanisms, it is possible that other pro-apoptotic gene products are upregulated by IFNxcex3, or anti-apoptotic gene products are downregulated, thus priming the cells for programmed cell death following a variety of apoptotic stimuli.
The ability to manipulate the mechanism by which the genes involved in cell death are regulated would provide physicians with a potential target for therapies aimed at ameliorating the effects of diseases that are characterized by abnormal levels of cell death and also would allow for the development of methods to identify agents that can effectively regulate, for example, apoptosis in a cell. However, the mechanisms by which these genes are regulated in a cell have not yet been fully elucidated. Thus, there exists a need to identify methods to manipulate the regulatory elements for genes involved in apoptosis. The present invention satisfies this need and provides related advantages as well.
All references cited herein, including all U.S. or foreign patents or are hereby incorporated by reference in their entirety.
The present invention provides nucleotide sequences that are gene regulatory elements, which regulate the expression of genes involved in cell death. The invention also provides the bak promotor, which regulates expression of the bak gene or heterologous genes linked to the bak promotor. The invention further provides screening assays for identifying agents such as drugs that effectively modulate expression of a gene that is controlled by a bak promoter and is involved in cell death. The invention also provides methods for modulating the level of apoptosis in a cell, and, specifically, in a mammal.
The present invention encompasses isolated polynucleotides comprising the bak promoter (SEQ ID NO:1) or an active fragment thereof. In one embodiment, the active fragment is positions xe2x88x921077 to xe2x88x921055 (positions 2945-2967 of SEQ ID NO:1), xe2x88x922376 to xe2x88x922360 (positions 1646-1662 of SEQ ID NO:1), xe2x88x922984 to xe2x88x922975 (positions 1038-1047 of SEQ ID NO:1), xe2x88x923073 to xe2x88x923064 (positions 949-958 of SEQ ID NO:1), or xe2x88x922591 to xe2x88x922549 (positions 1431-1473 of SEQ ID NO:1), of said bak promoter. The present invention encompasses methods for identifying an agent that effectively regulates, increases or decreases, the expression of a gene involved in apoptosis in a cell, comprising the steps of: a) introducing into said cell an isolated polynucleotide comprising a bak promoter or an active fragment thereof and a reporter gene; b) determining the level of expression of said reporter gene in said cell of step (a); c) contacting said cell of step (a) with the agent; and d) identifying an effective agent that regulates, increases or decreases, the expression of said reporter gene. Also encompassed is the effective agent identified according to the above method.
The present invention also encompasses methods of reducing or inhibiting the level of apoptosis in a cell, and specifically, methods of treating a patient having a disease characterized by an abnormal level of apoptosis, comprising administering to the cell or patient the effective agent identified above.
The invention also encompasses methods of reducing or preventing toxicity of a normal cell in a patient receiving therapy, including, but not limited to chemotherapy or radiotherapy, comprising administering to the patient a pharmaceutically acceptable composition comprising the effective agent identified above.